Polycyclic compounds and processes for the preparation thereof

ABSTRACT

N-(tetrahydropyran-4-yloxy-alkanoyl)-staurosporin derivatives of formula I ##STR1## wherein R 1  is hydrogen, hydroxy, lower alkoxy or oxo, 
     R 2  is hydrogen or C 1-4  alkyl and 
     R 3  is hydrogen or C 1-4  alkyl, 
     processes and novel intermediates for the preparation thereof and processes for the preparation of the intermediates are described. The compounds of formula I inhibit the enzyme protein kinase C with a high degree of selectivity and can be used especially as tumour-inhibiting active ingredients.

This is a divisional of Ser. No. 234,763, filed Apr. 28, 1994, now U.S.Pat. No. 5,382,675.

The invention relates to N-acylated staurosporin derivatives, namelyN-(tetrahydropyran-4-yloxy-alkanoyl)-staurosporin derivatives, toprocess and intermediate, for the preparations, thereof, topharmaceutical compositions comprising those compounds, to the usethereof as medicaments and to processes for the preparation of theintermediates.

Staurosporin, the basic element of the derivatives according to thepresent invention was isolated already in the year 1977 from cultures ofStreptomyces staurosporeus AWAYA, TAKAITASHI and OMURA, sp. nov. AM2282, cf. S. Omura et. al., J. Antibiot. 30, 275-281 (1977). Hitherto,only the relative, but not the absolute configuration of staurosporinwas known. The absolute configuration was published only recently by N.Funato et. al., Tetrahedron Letters 35:8, 1251-1254 (1994) andcorresponds to the mirror image of the structure, used up to now in theliterature to denote the rigid configuration of staurosporine.Accordingly, in the Tetrabedron Letters publication it is literallyrecommended "that the stereochemical notation for staurosporin which hasbeen in common use hitherto should be revised". Although the absoluteconfiguration was not known hitherto, it was unequivocally fixed(defined) by the designation as "staurosporin derivatives". Therefore,in order to avoid errors upon comparison with the priority applications,the original formulae are still used in the present application.

Staurosporin exhibits a strong inhibitory activity on protein kinase Cbut also inhibits other protein gases to an equally great extent andtherefore does not have the selectivity required for therapeutic use.Although staurospofin derivatives substituted by customary. acylradicals, such as benzoyl, are more selective, those N-acylatedstaurosporin derivatives are generally comparatively poorly soluble and,therefore, cannot easily be formulated into suitable pharmaceuticaldosage forms.

The aim of the present invention was to provide novel staurosporinderivatives that, whilst retaining the inhibitory activity ofstaurosporin on protein kinase C (PKC), especially on the "conventional"isotypes α, β-1, β-2 and γ of protein kinase C, principally on PKC-α andPKC-γ, are substantially less active in respect of other protein kinasesand other isotypes of protein kinase C. In addition, the staurosporinderivatives to be provided should be highly active when administeredorally and sufficiently soluble to be formulated into suitablepharmaceutical dosage forms without any great difficulty.

The invention relates especially toN-(tetrahydropymn-4-yloxy-alkanoyl)-staurosporin derivatives of formulaI ##STR2## wherein R₁ is hydrogen, hydroxy, lower alkoxy or oxo,

R₂ is hydrogen or C₁₋₄ alkyl and

R₃ is C₁₋₄ alkyl or preferably hydrogen, to processes and intermediatesfor the preparation thereof, to pharmaceutical compositions comprisingthose compounds, to the use thereof as medicaments and to processes forthe preparation of the intermediates.

The configurations evident from formula I are intended to designate onlythe relative, but not the absolute stereochemistry. As outlined above,the absolute stereochemistry is probably shown by the following formulaIa. ##STR3##

The configuration at the C-R₂ atom is (D) or (L), preferably (D).

Lower alkoxy R₁ is C₁ -C₇ alkoxy, preferably C₁₋₄ alkoxy, especiallymethoxy.

C₁₋₄ alkyl R₂ or R₃ is preferably methyl.

The compounds of formula I exhibit valuable pharmacological properties:for example, they inhibit the enzyme protein kinase C with a high degreeof selectivity. Phospholipid-and calcium-dependent protein kinase Coccurs in cells in several forms and participates in various fundamentalprocesses, such as signal transmission, proliferation anddifferentiation, and also the release of hormones and neurotransmitters.The activation of that enzyme is effected either by receptor-mediatedhydrolysis of phospholipids of the cell membrane or by directinteraction with certain tumour-promoting active substances. Thesensitivity of the cell to receptor-mediated signal transmission can besubstantially influenced by modifying the activity of protein kinase C(as a signal transmitter). Compounds that are capable of influencing theactivity of protein kinase C can be used as tumour-inhibiting,anti-inflammatory, immunomodulating and antibacterial active ingredientsand may even be of value as agents against athemsclerosis and disordersof the cardiovascular system and central nervous system.

Porcine brain protein kinase C purified in accordance with the proceduredescribed by T. Uchida and C. R. Filburn in J. Biol. Chem. 259, 12311-4(1984) is used to determine the inhibitory activity on protein kinase C.The inhibitory activity of the compounds of formula I on protein kinaseC is determined in accordance with the procedure of D. Fabbro et. al.,Arch. Biochem. Biophys. 239, 102-111 (1985). In that test, the compoundsof formula I inhibit protein kinase C at an IC₅₀ of as low asapproximately from 0.01 to 0.05 μmol/liter. In contrast, the compoundsof formula I inhibit other enzymes, for example protein kinase A andtyrosine protein kinase, only at a much greater concentration, forexample a concentration 100 times greater, which demonstrates theselectivity of the compounds of formula I.

The porcine brain protein kinase C used in the above test is a mixtureof various sub-types (isotypes) of protein kinase C. If pure recombinantisotypes are used in the above test instead of porcine brain proteinkinase C it is found that the compounds of formula I inhibit the"conventional" isotypes α, β-1, β-2 and γ preferentially whereas the"non-conventional" isotypes δ, εand η and the "atypical" isoform ζ areinhibited to a distinctly lesser extent and in some cases hardly at all.

Recombinant PKC isotypes are cloned, expressed and purified in thefollowing manner:

The production of various proteins with the aid of baculoviruses, andtheir cloning and isolation from Sf9 insect cells are carried out asdescribed by M. D. Summers and G. E. Smith, "A manual method forbaculovirus vectors and insect cell culture procedure", Texas Agricul.Exptl. Station Bull. (1987), 1555. The construction and isolation ofrecombinant viruses for the expression of PKC-α (bovine), PKC-β1(human), PKC-β2 (human) and PKC-γ (human/bovine hybrid) in Sf9 cells areeffected in the manner described by Stabel et. al. [S. Stabel, M.Liyanage and D. Frith, "Expression of protein kinase C isozymes ininsect cells and isolation of recombinant proteins", Meth. Neurosc.(1993)]. The production of the PKC isotypes in Sf9 cells is carried outin the manner indicated by Stabel et. al. (see above), and thepurification of the enzymes is effected in accordance with the methoddescribed in the publication by MeGlynn et. al. [E. McGlynn, J.Liebetanz, S. Reutener, J. Wood, N. B. Lydon, H. Hofstetter, M. Vanek,T. Meyer and D. Fabbro, " Expression and partial characterization of ratprotein kinase C-δ and protein kinase C-ζ in insect cells usingrecombinant baculovirus", J. Cell. Biochem. 49, 239-250 (1992)]. For thegeneration of recombinant PKC-δ(rat), PKC-ε (rat), PKC-ζ (rat) and PKC-η(mouse), and their expression and purification, the procedure describedby Liyanage et. al. ["Protein kinase C group B members PKC-δ,-ε,-ζ andPKC-λ: Comparison of properties of recombinant proteins in vitro and invivo", Biochem. J. 283, 781-787 (1992)]and McGlynn et. al.,respectively, (see above) is followed, with the additional feature thatthe transfer vector pAc360 is used for the expression of PKC-η [V.Luckow and M. D. Summers, "Trends in the development of baculovirusexpression", Biotechnology 6, 47-55 (1988)].

The measurement of the activity of the recombinant PKC isotypes obtainedby the above method is carried out in the absence of lipid and calcium(co-factors). Protamine sulfate phosphorylated in the absence ofco-factors is used as the substrate. The activity of the enzymesreflects the transfer of ³² P from γ-[³² P]-ATP to protamine sulfate.Protamine sulfate is a mixture of polypeptides each comprising fourC-terminal arginine residues. Phosphate incorporation is measured underthe following conditions: 100 μl of the reaction mixture contain infinal concentrations 20 mM TRIS-HCl pH 7.4, 10 mM Mg[NO₃ ]₂, 0.5 mg/mlof protamine sulfate, 10 μM ATP (0.1 μCi γ-[³² P]-ATP; 10 Ci/mol;Amersham. Little Chalfont. United Kingdom), various concentrations ofthe inhibitory compounds and 0.5-2.5 U (units: a unit is the amount ofenzyme that, in one minute and per milligram of protein, transfers onenanomole of ³² P from the above-mentioned γ-[³² p]-ATP to histone H1[Sigma, type V-S] ) of the enzymes. The reaction is started by theaddition of the enzymes and transfer at 32° C. The reaction time is 20minutes. The reaction is then stopped by dripping aliquots of 50 μl ontoP81 chromatography paper (Whatman, Maidstone, United Kingdom). Afterremoving unbound γ-[³² P]-ATP and nucleotide fragments by washingoperations as described by J. J. Witt and R. Roskoski, "Rapid proteinkinase assay using phospho-cellulose-paper absorption", Anal. Biochem.66, 253-258 (1975), the substrate phosphorylation is determined byscintillation measurement. In that test the compounds of formula Iinhibit the various isotypes-of protein kinase C (PKC) at an IC₅₀ of aslow as approximately from 0.001 to 0.1 μmol/liter in the case of PKC-αand PKC-γ, approximately from 0.01 to 0.08 ,μmol/liter in the case ofPKC-β-1 and PKC-β-2, approximately from 0.03 to 10 μmol/liter in thecase of PKC-δ,PKC-ε and PKC-η, and more than 4 μmol/liter in the case ofPKC-ζ.

As may be expected purely on the basis of the above-described inhibitoryactivity on protein kinase C, the compounds of formula I exhibitantiproliferative properties which can be demonstrated directly inanother test described in the following in which the inhibitory activityof the compounds of formula I on the growth of human T24 bladdercarcinoma cells is determined. Those cells are incubated in Eagle'sminimal essential medium, to which 5% (v/v) fetal calf serum has beenadded, in a humidified incubator at 37° C. and with 5% by volume of CO₂in the air. The carcinoma cells (1000-1500) are sown in 96-wellmicrotitre plates and incubated overnight under the above-mentionedconditions. The test compound is added in serial dilutions on day 1. Theplates are incubated for 5 days under the above-mentioned conditions.During that period the control cultures undergo at least four celldivisions. After incubation, the cells are fixed with 3.3% (w/v) aqueousglutaraldehyde solution, washed with water and stained with 0.05%(weight/volume) aqueous methylene blue solution. After washing, the dyeis eluted with 3% (w/v) aqueous hydrochloric acid. The optical density(OD) per well, which is directly proportional to the number of cells, ismeasured at 665 nm using a photometer (Titertek multiskan). The IC₅₀values are calculated with a computer system using the formula ##EQU1##

The IC₅₀ values are defined as being the concentration of activeingredient at which the number of cells per well is only 50% of thenumber of cells in the control cultures at the end of the incubationperiod. In the case of the compounds of formula I, the IC₅₀ values soobtained are approximately from 0.01 to 0.9 μmol/liter, especiallyapproximately from 0.03 to 0.9 μmol/liter.

The anti-tumour activity of the compounds of formula I can also bedemonstrated in vivo:

Female Balb/c hairless mice with s.c. transplanted human bladder tumoursT24 are used to determine the anti-tumour activity. With the animalsunder peroral forene narcosis, approximately 25 mg of a solid tumour areplaced under the skin on "the animals' left flank on day 0 and the smallincised wound is closed by means of suture clips. On day 6 after thetransplantation, the mice are divided at random into groups of 6 animalsand treatment commences. The treatment is carried out for 15 days withperoral or intraperitoneal administration once daily of a compound offormula I in dimethyl sulfoxide/Tween 80/sodium chloride solution in thevarious doses. The tumours are measured twice a week with a slide gaugeand the volume of the tumours is calculated. In this test, the peroralor intraperitoneal administration of 3 mg/kg daily of a compound offormula I brings about a reduction in the average tumour volume to from10 to 15% of the tumour volume in the untreated control animals.

On the basis of the properties described, the compounds of formula I canbe used especially as tumour-inhibiting active ingredients, for examplein the treatment of tumours of the bladder and the skin. When thecompounds of formula I are used in the treatment of cancer incombination with other chemotherapeutic agents, they prevent thedevelopment of resistance (multidrug resistance) or eliminate an alreadyexisting resistance to the other chemotherapeutic agents. They are alsosuitable for the other uses mentioned above for protein kinase Cmodulators and can be used especially in the treatment of disordersresponsive to inhibition of protein kinase C.

The compounds of formula I also inhibit certain tyrosine kinases, suchas, especially, PDGF receptor kinase, even at an IC₅₀ of less than 0.08μmol/liter.

PDGF (Platelet-derived Growth Factor) is a very frequently occurringgrowth factor which plays an important part both in normal growth and inpathological cell proliferation, such as in carcinogenesis and disordersof the smooth muscle cells of blood vessels, for example inatherosclerosis and thrombosis.

The inhibition of protein kinase C and PDGF receptor kinase acts in thatrespect quasi synergistically in the same way with a view to regulatingcell growth.

The inhibition of PDGF-stimulated receptor tyrosine kinase activity invitro is measured in PDGF receptor immune complexes of Balb/c 3T3 cellsin a manner analogous to that described by E. Andrejauskas-Buchdungerand U. Regenass in Cancer Research 52, 5353-5358 (1992). The compoundsof formula I described in more detail above inhibit PDGF-dependentcell-free receptor phosphorylation at concentrations of less than 0.08μmol/liter.

The inhibition of PDGF receptor tyrosine kinase in the intact cell isdemonstrated by Western blot analysis, likewise in a manner analogous tothat described by E. Andrejaus-kas-Buchdunger and U. Regenass in CancerResearch 52 the inhibition of the ligand-stimulated PDGF receptorautophosphorylation in Balb/c murine cells is measured by means ofanti-phosphotyrosine antibodies. The compounds of formula I inhibit thetyrosine kinase activity of the PDGF receptor at concentrations of from0.005 to 0.08 μmol/liter. Those compounds also inhibit the cell growthof a PDGF-dependent cell line, namely BALB/c 3T3 murine fibroblasts, atconcentrations of less than 1.0 μmol/liter.

On the basis of the properties described, the compounds of formula I canbe used not only as tumour-inhibiting active ingredients but also asagents against non-malignant proliferative disorders, such asatherosclerosis, thrombosis, psofiasis, sclerodermia and fibrosis. Theyare also suitable for the other uses mentioned above for protein kinaseC molulators and can be used especially in the treatment of disordersresponsive to inhibition of PDGF receptor kinase.

Compounds of formula I wherein R₁ is hydrogen or oxo, R₂ is hydrogen orC₁₋₄ alkyl and R₃ is hydrogen are preferred.

Compounds of formula I wherein R₁ is hydrogen or oxo, R₂ is hydrogen ormethyl and R₃ is hydrogen are especially preferred.

The above-mentioned compounds of formula I that have the(D)-configuration at the C-R₂ atom are more especially preferred.

The compounds of formula I described in the Examples, especiallyN-[O-(tetrahydro-pyran-4-yl)-D-lactoyl]-staurosporin, are even moreespecially preferred.

The compounds of formula I are prepared in accordance with processesknown per se. The process according to the invention comprises acylatingan amine of formula II ##STR4##

wherein R₁ is as defined above with the proviso that a hydroxy grouprepresented by R₁ is if necessary protected by a readily removablehydroxy-protecting group, with a carboxylic acid of formula III ##STR5##

wherein R₂ and R₃ are as defined above, or with a reactive carboxylicacid derivative thereof, and removing protecting groups, which are notpresent in the desired end product of formula I, and, if desired,separating a resulting mixture of isomers.

The manner in which the above-mentioned process is carried out isexplained in more detail hereinafter: protecting groups and the mannerin which they are introduced and removed are described, for example, in"Protective Groups in Organic Chemistry", Plenum Press, London, N.Y.1973, and in "Methoden der organischen Chemie", Houben-Weyl, 4thedition, Vol. 15/1, Georg-Thieme-Verlag, Stuttgart 1974 and in TheodoraW. Greene, "Protective Groups in Organic Synthesis", John Wiley & Sons,New York 1981. A characteristic of protecting groups is that they can bereadily removed, that is to say, without undesired secondary reactionstaking place, for example by solvolysis, reduction, photolysis or alsounder physiological conditions.

Hydroxy-protecting groups are, for example, acyl radicals, such asunsubstituted or substituted, for example halogen-substituted, loweralkanoyl, such as 2,2-dichloroacetyl, or acyl radicals of carbonic acidsemesters, especially tert-butoxycarbonyl-unsubstituted or substitutedbenzyloxycarbonyl, for example 4-nitrobenzyloxycarbonyl, ordiphenyl-methoxycarbonyl, or 2-halo-lower alkoxycarbonyl, such as2,2,2-trichloroethoxycarbonyl, and also trityl or formyl, or organicsilyl or stannyl radicals, and also readily removable etherflyinggroups, such as ten-lower alkyl, for example tert-butyl, 2-oxa- or2-thia-aliphatic or -cycloaliphafic hydrocarbon radi 1-loweralkylthio-lower alkyl, for example methoxymethyl, 1-methoxyethyl,1-ethoxy-ethyl, methylthiomethyl, 1-methylthioethyl or 1-ethylthioethyl,or 2-oxa- or 2-thia-cyclo-alkyl having 5 or 6 ring atoms, for exampletetrahydrofuryl or 2-tetrahydropyranyl or corresponding thia analogues,and also unsubstituted or substituted 1-phenyl-lower alkyl, such asunsubstituted or substituted benzyl or diphenylmethyl, suitablesubsfituents of the phenyl radicals being, for example, halogen, such aschlorine, lower alkoxy, such as methoxy, and/or nitro.

The removal of the protecting groups, which are not constituents of thedesired end product of formula I, is carried out in a manner known perse, for example by solvolysis, especially hydrolysis, alcoholysis oracidolysis, or by reduction, especially hydrogenolysis or chemicalreduction. Hydroxy protected by unsubstituted or substituted1-phenyl-lower alkyl, for example benzyl, is freed preferably bycatalytic hydrogenation, for example in the presence of apalladium-on-carbon catalyst. A hydroxy group protected by2,2-dichloroacetyl is freed, for example, by basic hydrolysis, and ahydroxy group etherified by tert-lower alkyl or by a 2-oxa- or2-thia-aliphatic or -cycloaliphatic hydrocarbon radical is freed byacidolysis, for example by treatment with a mineral acid or a strongcarboxylic acid, for example sulifluoroacetic acid. Hydroxy etherifiedby an organic silyl radical, for example trimethylsilyl, can also befreed by a hydrofluoric acid salt yielding fluoride anions, for exampletetrabutylammonium fluoride.

A reactive acid derivative of a compound of formula III is especially areactive (activated) ester, a reactive anhydride or a reactive cyclicamide.

Reactive (activated) esters of an acid of formula III are especiallyesters unsaturated at the linking carbon atom of the esterflyingradical, especially of the vinyl ester type, such as vinyl estersthemselves (which can be obtained, for example, by transesterifying acorresponding ester with vinyl acetate; activated vinyl ester method),carbamoyl vinyl esters (which can be obtained, for example, by treatingthe corresponding acid with an isoxazolium reagent; 1,2-oxazolium orWoodward method), or 1-lower alkoxyvinyl esters (which can be obtained,for example, by treating the corresponding acid with a loweralkoxyacetylene; ethoxyacetylene method), or esters of the amidino type,such as N,N'-di-substituted amidino esters (which can be obtained, forexample, by treating the corresponding acid with a suitableN,N'-di-substituted carbodiimide, for exampleN,N'-dicyclo-hexylcarbodiimide orN-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride;carbodiimide method), or N,N-di-substituted amidino esters (which can beobtained, for example, by treating the corresponding acid with anN,N-di-substituted cyanamide; cyanamide method), suitable aryl esters,especially phenyl esters suitably substituted by electron-attractingsubstituents (which can be obtained, for example, by treating thecorresponding acid with a suitably substituted phenol, for example4-nitrophenol, 4-methylsulfonylphenol, 2,4,5-trichlorophenol,2,3,4,5,6-pentachlorophenol or 4-phenyl-diazophenol, in the presence ofa condensation agent, such as N,N'-dicyclohexylcarbodiimide; activatedaryl esters method), cyanomethyl esters (which can be obtained, forexample, by treating the corresponding acid with chloroacetonitrile inthe presence of a base; cyanomethyl esters method), thio esters,especially unsubstituted or substituted, for example nitro-substituted,phenylthio esters (which can be obtained, for example, by treating thecorresponding acid with unsubstituted or substituted, for examplenitro-substituted, thiophenols, inter alia. by the anhydride orcarbodiimide method; activated thiol esters method), amino or amidoesters (which can be obtained, for example, by treating thecorresponding acid with an N-hydroxy-amino or N'-hydroxy-amino compound,for example N-hydroxy-succinimide, N-hydroxy-piperidine,N-hydroxy-phthaiimide or 1-hydroxy-benzotriazole, for example by theanhydride or carbodiimide method; activated N-hydroxy esters method) orsilyl esters (which can be obtained, for example, by treating thecorresponding acid with a silylating agent, for examplehexamethyldisilazane, and which react readily with hydroxy groups butnot with amino groups).

Anhydrides of an acid of formula HI may be symmetric or preferably mixedanhydrides of those acids, for example anhydrides with inorganic acids,such as acid halides, especially acid chlorides (which can be obtained,for example, by treating the corresponding acid with thionyl chloride,phosphorus pentachloride or oxalyl chloride; acid chloride method),azides (which can be obtained, for example, from a corresponding acidester by way of the corresponding hydrazide and treatment thereof withnitrous acid; azide method), anhydrides with carbonic acidsemi-derivatives, such as with corresponding esters, for examplecarbonic acid lower alkyl semiesters (which can be obtained, forexample, by treating the corresponding acid with haloformic acid loweralkyl esters, such as chloroformic acid lower alkyl esters, or with a1-lower alkoxycarbonyl-2-lower alkoxy-1,2-dihydroquinoline, for example1-lower alkoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; mixedO-alkylcarbonic acid anhydrides method), or anhydrides withdihalogenated, especially dichlorinated, phosphoric acid (which can beobtained, for example, by treating the corresponding acid withphosphorus oxychloride; phosphorus oxychloride method), or anhydrideswith organic acids, such as mixed anhydrides with organic carboxylicacids (which can be obtained, for example, by treating the correspondingacid with an unsubstituted or substituted lower alkane- or phenyl-loweralkane-carboxylic acid halide, for example phenylacetic acid chloride,pivaiic acid chloride or trifluoroacetic acid chloride; mixed carboxylicacid anhydrides method) or with organic sulfonic acids (which can beobtained, for example, by treating a salt, such as an alkali metal salt,of the corresponding acid with a suitable organic sulfonic acid halide,such as a lower alkane- or aryl-sulfonic acid chloride, for examplemethane- or p-toluene-sulfonic acid chloride; mixed sulfonic acidanhydrides method) and symmetric anhydrides (which can be obtained, forexample, by condensing the corresponding acid in the presence of acarbodiimide or 1-diethyl-aminopropyne; symmetric anhydrides method).

Suitable cyclic amides are especially amides having five-membereddiazacycles of aromatic character, such as amides with imidazoles, forexample imidazole (which can be obtained, for example, by treating thecorresponding acid with N,N'-carbonyldiimidazole; imidazolide method),or pyrazoles, for example 3,5-dimethylpyrazole (which can be obtained,for example, by way of the acid hydrazide by treatment withacetylacetone; pyrazolide method).

Derivatives of acids of formula III that are used as acylating agentscan also be formed in situ. For example, N,N'-di-substituted aminoesters can be formed in situ by reacting a mixture of the startingmaterial of formula V and the acid used as acylating agent in thepresence of a suitable N,N-di-substituted carbodiimide, for exampleN,N'-dicyclohexyl-carbodiimide. In addition, amino or amino esters ofthe acids used as acylating agents can be formed in the presence of thestarting material of formula V to be acylated, by reacting a mixture ofthe corresponding acid and amino starting materials in the presence ofan N,N'-di-substituted carbodiimide, for exampleN,N'-dicyclohexylcarbodiimide, and of an N-hydroxy-amine orN-hydroxy-amide, for example N-hydroxysuccinimide, where appropriate inthe presence of a suitable base, for example 4-dimethylaminopyridine.

The reaction can be carried out in a manner known per se, the reactionconditions depending especially upon whether and how the carboxy groupof the acylating agent of formula III has been activated, generally inthe presence of a suitable solvent or diluent or a mixture thereof, and,if necessary, in the presence of a condensation agent which, for examplewhen the carboxy group participating in the reaction is in the form ofan anhydride, may also be an acid-binding agent, with cooling orheating, for example in a temperature range of from approximately -30°C. to approximately +150° C., especially approximately from 0° C. to+100° C., preferably from room temperature (approximately +20° C.) to+70° C., in an open or closed reaction vessel and/or in the atmosphereof an inert gas, for example nitrogen. Customary condensation agentsare, for example, carbodiimides, for example N,N'-diethyl-,N,N'-dipropyl- or N,N'-dicyclohexyl-carbodiimide, suitable carbonylcompounds, for example carbonyldiimidazole, or 1,2-oxazolium compounds,for example 2-ethyl-5-phenyl-1,2-oxazolium-3'-sulfonate and2-tert-butyl-5-methyl-isoxazolium perchlorate, or a suitable acylaminecompound, for example 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline.Water-soluble carbodiimides, such asN-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide, are advantageous.Customary acid-binding condensation agents are, for example, alkalimetal carbonates or hydrogen carbonates, for example sodium or potassiumcarbonate or hydrogen carbonate (customarily together with a sulfate),or organic bases, such as customarily sterically hindered tri-loweralkylamines, for example N,N-diisopropyl-N-ethylamine.

Mixtures of isomers can be separated into the individual isomers in amanner known per. se, for example by fractional crystallisation,chromatography etc..

The starting materials of formula II are known or can be prepared inaccordance with processes known per se. The starting material of formulaII wherein R₁ is hydrogen, that is to say, staurosporin, is commerciallyavailable and can be prepared by fermentation with the strainStreptomyces staurosporeus. That strain was deposited under number FERMP-3725 at the Fermentation Research Institute, Japan, in connection withthe examined Japanese patent publication, Kokoku, No. 57-53 076 whichwas published on 11.11.1982, see S. Omura et. al., J. Antibiot. 30,275-281 (1977). Staurospofin derivatives of formula II wherein R₁ isother than hydrogen are described, for example, by I. Takahashi et. al.,J. Pharmacol. Exp. Ther. 255 (3) (1990) 1218-1221 and in WO-A- 8907 -105 -A (Applicant: Kyowa Hakko Kogyo KK, Japanese priority No. 024 571of 4.2.1988). Compounds of formula I wherein R₁ is hydroxy or oxo arealso obtained as secondary products in the synthesis of compounds offormula I wherein R₁ is hydrogen.

The starting materials of formula Ill are novel. An acid of formula IIIis obtained by reacting tetrahydropyran-4-oil with an acid of formula IV##STR6##

wherein X is a nucleophilic leaving group and R₂ and R₃ are as definedabove. A nucleophilie leaving group X is especially hydroxy estefifiedby a suitable mineral acid, such as a suitable hydrohalic acid, or asuitable sulfonic acid, such as 4-toluenesulfonic acid, preferablychlorine. Tetrahydropyran-4-oil is first reacted in a suitable inertaprotic solvent, such as an acyclic or cyclic ether, such as dioxane,with a suitable base, such as sodium hydride. The resulting suspensionis added dropwise to a solution of a compound of formula IV in asuitable inert apmtic solvent, such as an acyclic or cyclic ether, suchas dioxane. The reaction is carried out at from 0° C. to 150° C.,preferably from 20° C. to 100° C., for example at the reflux temperatureof the solvent used.

The invention relates also to the novel compounds of formula 1II andtheir salts as intermediates for the preparation of the compounds offormula I. The compounds of formula 1II are surprisingly soluble inwater and organic solvents. The water solubility at 22° C. is from 100 gto 500 g/liter. It is therefore possible that the corresponding acylradicals of the compounds of formula III are largely responsible for thesubstantially increased solubility, for example increased more than10-fold, of the compounds of formula I in water and other solvents, bycomparison with that of other N-acyl-staurosporin derivatives, such asN-benzoyl- staurosporin.

Preferred are compounds of formula III wherein R₂ is hydrogen or methyland R₃ is hydrogen or methyl, especially the compounds of formula 1IIdescribed in the Examples and the salts thereof.

Salts of compounds of formula Ill are especially metal or ammoniumsalts, such as alkali metal or alkaline earth metal salts, for examplesodium, potassium, magnesium or calcium salts, or ammonium salts withammonia or with suitable organic amines, such as tertiary monoamines,for example methylamine or tri-(2-hydroxyethyl)-amine, or heterocyclicbases, for example N-ethyl-pipefidine or N,N'-dimethyl-piperazine.

The invention relates also to the process described above for thepreparation of the novel compounds of formula III.

The processes described above, including the processes for removingprotecting groups and the additional process measures are, unlessotherwise indicated, carried out in a manner known per se, for examplein the presence or absence of preferably inert solvents or diluents, ifnecessary in the presence of condensation agents or catalysts, atreduced or elevated temperature, for example in a temperature range offrom approximately -20° C. to approximately 150° C., especially fromapproximately 0° C. to approximately +70° C., preferably fromapproximately +10° C. to approximately +50° C., principally at roomtemperature, in a suitable vessel and, if necessary, in an inert gasatmosphere, for example a nitrogen atmosphere.

Taking into account all the substituents in the molecule, if necessary,for example if readily hydrolysable radicals are present, especiallymild reaction conditions are to be used, such as short reaction times,the use of mild acidic or basic agents in low concentration,stoichiometric ratios, and the selection of suitable catalysts,solvents, temperature conditions and/or pressure conditions. Theinvention relates also to the use of the compounds of formula I,preferably in the form of pharmaceutical compositions, in thetherapeutic treatment of the human or animal body, especially in thecase of the abovementioned disorders. The invention relates also to amethod of inhibiting protein kinase C in a warm-blooded animal requiringsuch treatment, which comprises administering to that warm-bloodedanimal a dose that is effective in inhibiting protein kinase C of acompound of formula I. The dose of the active ingredient depends, interalia, on the nature of the disorder, the type and size of the species tobe treated, the organism's resistance and the mode of administration.For example, a warm-blooded animal of approximately 70 kg body weightreceives a daily dose of from 1 mg to 1500 mg, principally from 1100 mgto 1000 mg, preferably from 200 mg to 800 mg, for example 500 mg, of acompound of formula I. That total daily dose is preferably divided into2 or 3 administrations daily. The dose for oral administration isapproximately from two to three times greater than for parenteraladministration, that is to say, it tends towards the upper range of thedoses indicated.

The invention relates also to pharmaceutical compositions comprising aneffective amount, especially an amount effective in the prophylaxis ortreatment of one of the above-mentioned disorders, of the activeingredient together with pharmaceutically acceptable carders that aresuitable for topical, enteral, for example oral or rectal, or parenteraladministration and that may be inorganic or organic, solid or liquid.There are used for oral administration especially tablets or gelatincapsules that comprise the active ingredient together with diluents, forexample lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/orglycerol, and/or lubricants, for example silica, talc, stearic acid orsalts thereof, such as magnesium or calcium stearate, and/orpolyethylene glycol. Tablets may also comprise binders, for examplemagnesium aluminium silicate, starches, such as corn, wheat or ricestarch, gelatin, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, and, if desired, disintegrators, for examplestarches, agar, alginic acid or a salt thereof, such as sodium alginate,and/or effervescent mixtures, or adsorbents, dyes, flavourings andsweeteners. It is also possible to use the pharmacologically activecompounds of the present invention in the form of parenterallyadministrable compositions or in the form of infusion solutions. Suchsolutions are preferably isotonic aqueous solutions or suspensionswhich, for example in the case of lyophilised compositions that comprisethe active ingredient alone or together with a carrier, for examplemannitol, can be made up prior to use. The pharmaceutical compositionsmay be sterilised and/or may comprise excipients, for examplepreservatives, stabilisers, wetting agents and/or emulsifiers,solubilisers, salts for regulating the osmotic pressure and/or buffers.The present pharmaceutical compositions, which may, if desired, compriseother pharmacologically active substances, such as antibiotics, areprepared in a manner known per se, for example by means of conventionalmixing, granulating, confectioning, dissolving or lyophilisingprocesses, and comprise approximately from 0.01% to 90%, in the case oflyophilisates up to 100%, especially from approximately 0.1% toapproximately 50%, especially from 1% to 30%, of the activeingredient(s), an active ingredient concentration of less than 1% beingsuitable especially for compositions that are to be applied topically.

The following Examples illustrate the invention without limiting it inany way. The R_(f) values are determined on silica gel thin-layer plates(Merck, Darmstadt, Gemany). The ratio of the eluants in the eluantmixtures used is indicated in pans by volume (v/v) and temperatures areindicated in degrees Celsius. The concentration, c, of the compound inthe solvent or solvent mixture is, in the case of the optical rotation,indicated as a percentage (weight/volume).

Example 1: At 0°, 1.17 g (7.8 mmol) of 1-hydroxybenzotriazole and 1.61 g(7.8 mmol) of N,N'-dicyclohexylcarbodiimide are added to a solution of1.13 g (6.5 mmol) of O-tetra-hydropyran-4-yl-D-lactic acid in 75 ml ofabsolute N,N'-dimethylformamide and the resulting clear colourlesssolution is stirred for 3 hours at 0°.2.33 g (5.01 mmol) of staurospofinare then added and the resulting colourless suspension is stirred for 1hour at 0° and for 20 hours at room temperature. Then, in order toensure that the staurospofin used has been completely reacted, an activeester solution of 0.38 g (2.18 mmol) of 0-tetrahydro-pyran-4-yl-D-lacticacid, 0.39 g (2.60 mmol) of 1-hydroxybenzotrizole and 0.54 g (2.60 mmol)of N,N'-dicyclohexylcarbodiimide in a total of 25 ml of absoluteN,N'-dimethylformamide is added again and the batch is stirred for onehour at 0° and then for a further 18 hours at room temperature. Theresulting yellowish suspension is poured into 300 ml of water andstirred for one hour at room temperature and the precipitated crystalsare filtered off with suction and washed with water. The aqueous phaseis discarded. The filter material is suspended in 130 ml of methylenechloride and stirred for 1.5 hours at room temperature. The precipitatedN,N'-dicyclohexylurea is filtered off with suction and washed withmethylene chloride and the filtrate is concentrated to dryness byevaporation under a high vacuum at 30° . The residue (yellow crystals)is purified by column chromatography on 300 g of silica gel (type Si60,Merck 9385, 0.040-0.063 mm) in chloroform (25 ml fractions). Fractions220-305 are combined and concentrated to dryness by evaporation under ahigh vacuum at 30° . The residue (yellow crystals) is recrystallisedtwice from ethyl acetate to giveN-[O-(tetrahydropyran-4-yl)-D-lactoyl]-staurosporin in the form ofslightly yellowish crystals of m.p. 222°-223° (sintering from 220° )which still contain 0.19 mol of water, [α]²⁰ _(D) =+166.9±2.0°(c=0.498;methanol).

The starting material is obtained as follows:

Stage 1.1:4.8 g (120 mmol) of 60% sodium hydride in oil (Fluka, pract.)are added at 65° to a solution of 3.06 g (2.85 ml, d=1.074; 29.96 mmol)of tetrahydro-2H-pyran-4-oil (Fluka, pract.) in 100 ml of absolute1,4-dioxane. The resulting grey suspension is stirred under reflux for 2hours, is allowed to cool to 65° again and then a solution of 3.25 g(2.59 ml, d=1.25; 29.95 mmol) of S(-)-2-chloropropionic acid (Fluka,puriss.) in 60 ml of absolute 1,4-dioxane is added dropwise over aperiod of 8 minutes. The resulting brown suspension is diluted with 100ml of absolute 1,4-dioxane and the batch is heated under reflux for 3hours, with stirring. Stirring is then continued for a further 14 hoursat room temperature. 40 ml of water are then added dropwise to theresulting brown suspension over a period of 2 minutes and the yellowsolution obtained is concentrated to dryness by evaporation under a highvacuum. The residue is taken up in 200 ml of water and the aqueoussolution is extracted once with 250 ml and once with 150 ml of ethylacetate. The ethyl acetate extracts are then washed once with 100 ml ofwater. All the aqueous phases are combined and then acidified with 4Nhydrochloric acid (pH 1 ). The resulting solution is saturated withsodium chloride and extracted twice with 300 ml of ethyl acetate eachtime. The organic phases are then washed three times with 150 ml ofsaturated sodium chloride solution each time. All the ethyl acetateextracts are then combined, dried over magnesium sulfate, filtered andconcentrated to dryness by evaporation under a high vacuum at 30° . Theresidue (yellow oil) is purified by bulb tube distillation (b.p.approximately 160° at 0.6 mm Hg). O-(tetrahydropyran-4-yl)-D-lactic acidis obtained in the form of a slightly yellowish oil; [α[²⁰ _(D)=+46.7±0.9+ (c=1.058;chloroform). The oil crystallises from ethylacetate/hexane 1:1 in the form of colourless crystals having a meltingpoint of 68.7°-69.5°[α]_(D) ²⁰ =+48.8±0.8°(c=1;chloroform).

Example 2: From the mother liquors of the recrystallisations of the endproduct of Example 1 there is isolated by flash chromatography at 0.4bar on 90 g of silica gel (type Si60, Merck 9385, 0.040-0.063 mm) inmethylene chloride/methanol (98:2; 10 ml fractions), after concentratingfractions 23-27 to dryness by evaporation under a high vacuum,N-[O-(tetrahydropyran-4-yl)-D-lactoyl]-7-oxo-staurospofin in the form ofyellow crystals of m.p. 206°-208° (without recrystallisation), (+)FAB,MS: (M+H)⁺ =637, [α]²⁰ _(D) =+138.7±10.8°(c=0.185;methanol:chloroform=1:1).

Example 3:9.16 g (61.0 mmol) of 1-hydroxybenzotriazole and 11.7 g (61.0mmol) of N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride(EDC) are added at 0° under argon to a solution of 8.16 g (46.9 mmol) ofO-tetrahydropyran-4-yl-D-lactic acid in 400 ml of absoluteN,N'-dimethylformamide and the resulting clear colourless solution isstirred for 3 hours at 0°.17.50 g (37.5 mmol) of staurospofin are thenadded and the resulting yellowish solution is stirred for 2 hours at 0°and for 19 hours at room temperature. The yellowish solution issubsequently concentrated to dryness by evaporation under a high vacuum.The residue is stirred for 15 minutes with 250 ml of water, the batch isfiltered with suction and the resulting beige crystals are washed withwater. The crystals are purified by flash chromatography at 0.4 bar on500 g of silica gel (type Si60, Merck 9385, 0.040-0.063 mm) in methylenechloride/methanol (98:2; 25 ml fractions). Fractions 70-140 are combinedand concentrated by evaporation under a high vacuum at 30° . The residueis crystallised from 400 ml of ethyl acetate, andN-[0-(tetrahydropyran-4-yl)-D-lactoyl]-staurosporin that is alm obtainedin the form of beige crystals. Fractions 45-69 and 141-170 of theabove-mentioned flash chromatography are likewise combined andconcentrated by evaporation under a high vacuum. The yellow crystals soobtained and the yellow crystals from the mother liquor of the firstrecrystallisation are subjected to flash chromatography again on 500 gof silica gel Si60 under the same conditions as those already described.After concentrating by evaporation the TLC-pure fractions of the secondflash chromatography operation, the products are combined with the beigecrystals first obtained and recrystallised again from 800 ml of ethylacetate to give N-[O -(tetrahydropyran-4-yl)-D-lactoyl]-staurospofin inthe form of beige crystals of m.p. 220°-222° (sintering from 214°) whichstill contain 0.42 mol of water; [α]D²⁰ =+166.6+2.5° (c=0.404;methanol).

Example 4: Analogously to Example 3, there is obtained from 310 mg (1.78mmol) of O-tetrahydropyran-4-yl-L-lactic acid, 347 mg (2.31 mmol) of1-hydroxybenzotriazole, 443 mg (2.31 mmol) ofN-ethyl-N'-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)and 664 mg (1.42 mmol) of staurosporin in 15 ml of absoluteN,N'-dimethylformamide, after a reaction time of 5 hours at 0° and 16hours at room temperature under argon and subsequent analogous flashchromatography, N-[O-(tetrahydro-pyran-4-yl)-L-lactoyl]-staurosporin inthe form of beige (sintering from 280° ; from ethyl acetate); [α]_(D) ²⁰=+145.6±1.8° (c=0.544;chloroform).

The starting material is

Stage 4.1; Analogously to stage 1.1, there is obtained from 1.021 g(0.951 ml, d =1.074; 10 mmol) of tetrahydro-2H-pyran-4-oil (Fluka,pract.), 1.60 g of sodium hydride (60% strength in oil, Fluka pract.)and 1.08 g (0.863 ml, d =1.258; 10 mmol) of R(+)-2-chloropropionic acid(Fluka, puriss.) in a total of 55 ml of 1,4-dioxane, after concentrationof the ethyl acetate extracts by evaporation and after bulb tubedistillation of the resulting residue (b.p. approximately 160° , at 0.8mm Hg), O-(tetrahydropyran-4-yl)-L-lactic acid in the form of acolourless oil which, on being left to stand, solidifies to colourlesscrystals which melt at from 33.7° to 67.6° and still contain 0.13 mol(1.30%) of water, [α]_(D) ²⁰ =-46.7±1.0° (c=1.035; chloroform).

Example 5: Analogously to Example 3 there is obtained from 200 mg (1.25mmol) of O-tetrahydropyran-4-yl-glycolic acid, 244 mg (1.62 mmol) of1-hydroxybenzotriazole, 311 mg(1.62 mmol) ofN-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and466 mg (1.0 mmol) of staurosporin in 10 ml of absoluteN,N'-dimethylformamide, after a reaction time of 2 hours at 0° and 18hours at room temperature, flash chromatography (analogously to Example3, but 20 ml fractions, product in fractions 15-20) andrecrystallisation of the product so purified from ethyl acetate/hexane(1:1), N-[2-(tetrahydropyran-4-yloxy)-acetyl]-staurosporin in the formof beige crystals of m.p. 222°-224° (sintering from 215° ) which stillcontain 0.29 mol of water; [α]_(D) ²⁰ =+180.0+2.0° (c=0.510;chloroform), R_(f) =0.26 (methylene chloride:ethanol=95:5); R_(f) =0.48(acetone); R_(f) =0.64 (methylene chloride:methanol=9:1).

The starting material is obtained as follows:

Stage 5.1: At 65°, 3.20 g (80 mmol) of 60% sodium hydride in oil(Fluka., pract.) are added to a solution of 2.042 g (1.902 ml, d=1.074;20.0 mmol) of tetrahydro-2H-pyran4-oil (Fluka, pract.) in 70 ml ofabsolute 1,4-dioxane. The resulting grey suspension is stirred for 3hours under reflux, is allowed to cool to 65° again and then a solutionof 1.89 g (20.0 mmol) of chloroacetic acid (Fluka, pufiss.) in 40 ml ofabsolute 1,4-dioxane is added dropwise over a period of 20 minutes. Theresulting grey-brown suspension is then heated to reflux again andstirred for 3 hours at that temperature. After stirring for a further 16hours at room temperature, 10 ml of water are added dropwise over aperiod of 5 minutes and the resulting yellowish suspension isconcentrated to dryness by evaporation under a high vacuum. The residueis taken up in 20 ml of water and the aqueous solution is extractedtwice with 20 ml of ethyl acetate each time. The ethyl acetate extractsare then washed once with 10 ml of water. The aqueous phases arecombined and then acidified with 4N hydrochloric acid (pH 1 ). Theresulting solution is saturated with sodium chloride and extracted twicewith 50 ml of ethyl acetate each time. The ethyl acetate phases are thenwashed twice with 20 ml of saturated sodium chloride solution each time.All the ethyl acetate extracts are subsequently combined, dried overmagnesium sulfate, filtered and concentrated to dryness by evaporationunder a high vacuum at 30° . The residue (yellow oil) is purified bybulb tube distillation (b.p. approximately 130° at 0.4 mm Hg) to give0-(tetrahydropyran-4-yl)-glycolic acid in the form of a colourless oilwhich, on being left to stand, solidifies to colourless crystals whichmelt at from 63.9 to 70.6° (sintering from 60.2° ) and contain 0.08 mol(0.91%) of water.

Example 6: From fractions 9-11 of the flash chromatography of Example 5there is obtained as a secondary product, after recrystailisation fromethyl acetate/hexane 1:1,N-[2-(tetrahydropyran-4-yloxy)-acetyl]-7-oxo-staurospofin in the form ofyellow crystals of melting point 190.7°-192.4° (sintering from 187° )which still contain 0.35 mol of water;, [α_(D) ²⁰ +157.4±2.0° (c=0.491;chloroform), R_(f) =0.35 (methylene chloride:ethanol=95:5), R_(f) =0.63(acetone), R_(f) =0.73 (methylene chloride:methanol=9.1).

Example 7: From fractions 26-36 of the flash chromatography of Example 5there is obtained as a secondary product which is unstable in solutionN-[2-(tetrahydropyran-4-yl-oxy)-acetyl]-7-hydroxy-staurospo crystals ofm.p. 235°-237° (sintering from 227° ; from ethyl acetate/hexane 1:1)which still contain 0.69 mol of water, [α]_(D) ²⁰ =+209.6±2.0° (c=0.125;chloroform), R_(f) =0.24 (methylene chloride:ethanol =95:5), R_(f) =0.55(acetone), Re ⁼ 0.55 (methylene chloride:methanol=9.1).

Example 8: Analogously to Example 3 there is obtained from 1.327 g (7.05mmol) of 2-methyl-2-(tetrahydropyran-4-yloxy)-propionic acid, 1.376 g(9.16 mmol) of 1-hydroxy-benzotriazole, 1.757 g (9.16 mmol) ofN-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and2.63 g (5.64 mmol) of staurosporin in 50 ml of absoluteN,N'-dimethylformamide, after a reaction time of 24 hours at roomtemperature under argon, flash chromatography at 0.4 bar (20 mlfractions) on 500 g of silica gel (type Si60, Merck 9385, 0.040-0.063mm) in methylene chloride/acetone (9:1; fractions 1-100) and methylenechloride/acetone (1:1; fractions 100-200),N-[2-methyl-2-(tetrahydropyran-4-yloxy)-propionyl]-staurosporin. Forfurther purification, fractions 134-170 are combined and concentrated todryness by evaporation under a high vacuum at 30° . The resultingresidue is purified again by flash chromatography at 0.4 bar on 100 g ofsilica gel (type Si60, Merck 9385, 0.040-0.063 mm) in methylenechloride/methanol (98:2; 25 ml fractions). Fractions 21-28 are combinedand concentrated by evaporation under a high vacuum at 30 ° .Recrystallisation of the residue from 13 ml of ethyl acetate/cyclohexane(1:4) givesN-[2-methyl-2-(tetrahydropyran-4-yloxy)-propionyl]-staurosporin in theform of slightly beige crystals of m.p. 209-211° (sintering from 204° )which still contain 0.38 mol (1.07%) of water; [α]_(D) ²⁰ =+154.7±2.0°(c=0.497; chloroform).

The starting material is obtained as follows:

Stage 8.1a: A solution of 3.48 g (20 mmol) ofO-(tetrahydropyran-4-yl)-D-lactic acid in 20 ml of absolutetetrahydrofuran is added dropwise under argon over a period of 15minutes at 0°-9° to a solution of 20 ml (40 mmol) of lithiumdiisopropylamide (2-molar solution in tetrahydrofuran/cyclohexane) in 20ml of absolute tetrahydrofuran. The resulting red solution is thenstirred for one hour at 0° and subsequently cooled to -75° . A solutionof 2.84 g (1.25 ml, d =2.280; 20 mmol) of methyl iodide in 10 ml ofabsolute tetrahydrofuran is then added dropwise over a period of 2minutes, during which time the temperature rises to -61° and the colourof the solution changes to yellow. The yellow solution is stirred for afurther 14 hours with gradual warming to room temperature and then theresulting yellow suspension is poured onto 100 ml of ice-water and thebatch is extracted twice with 100 ml of ethyl acetate each time. Theethyl acetate phases are then washed once with 50 ml of water. Theaqueous phases are combined, acidified with 4N hydrochloric acid andextracted twice with 100 ml of ethyl acetate each time. The ethylacetate phases are then washed twice with 50 ml of water each time,combined, dried over sodium sulfate and concentrated to dryness byevaporation under a high vacuum at 30°. The crude product is dissolvedin 30 ml of ethyl acetate, and 2.73 ml of dicyclohexylamine (Fluka,puriss.) are added to the resulting solution at room temperature. Theslowly precipitated crystals are filtered off with suction, washed witha small amount of ethyl acetate and recrystallised a further twice from30 ml of ethyl acetate each time to give the dicyclohexylammonium saltof 2-methyl-2-(tetrahydropyran-4-yloxy)-propionic acid in the form ofcolourless crystals which melt at from 131.7° to 137.8° (sintering from128° ).

The salt can be used directly for further synthesis or can be reacted asfollows to form the free 2-methyl-2-(tetrahydropyran-4-yloxy)-propionicacid. 3.8 g (0.01 mol) of the dicyclohexylammonium salt are dissolved in50 ml of water. The solution is adjusted to a pH of 1 with 4Nhydrochloric acid. The precipitated dicyclohexylammonium chloride isfiltered off with suction and washed with a small amount of water. Theaqueous phase is then extracted twice with 100 ml of ethyl acetate eachtime. The extracts are washed twice with 50 ml of water each time,combined, dried over sodium sulfate and concentrated to dryness byevaporation under a high vacuum at 30° . The residue is recrystallisedfrom cyclohexane to give 2-methyl-2-(tetrahydropyran-4-yloxy)-propionicacid in the form of colourless crystals which melt at from 90.2° to93.8° (sintering from 79.5° ).

Alternatively, 2-methyl-2-(tetrahydropyran-4-yloxy)-propionic acid canbe obtained in accordance with a process described by H. Gilman and G.R. Wilder in J. Am. Chem Soc. 77, 6644 (1955) in the following manner:

Stage 8.1b: 5.10 g (50 mmol) of tetrahydro-2H-pyran-4-oil (Fluka,pract.) are dissolved in 48.0 g (60 ml, d =0.79; 826 mmol) of absoluteacetone (Fluka, puriss.). 8.11 g (5.45 ml, d =1.49; 68 mmol) ofchloroform (Fluka, puriss.) and 9.60 g (240 mmol) of sodium hydroxide(Merck, p.a.) are gradually added to the solution at room temperature,with stirring, the temperature of the reaction mixture rising from 23°to 58° (reflux) over a period of 10 minutes. After 30 minutes at 58° theresulting colourless suspension slowly cools again spontaneously. It isheated to reflux again and stirred for a further 5 hours at thattemperature. After cooling again, the batch is concentrated to drynessby evaporation under a high vacuum at 30°. The residue is taken up in 50ml of water, acidified with 4N hydrochloric acid (pH 1) and extractedtwice with 100 ml of ethyl acetate each time. The extracts are washedtwice with 50 ml of saturated sodium chloride solution each time. Theethyl acetate phases are combined, dried over sodium sulfate, filteredand concentrated by evaporation again. The residue is purified by columnchromatography on 500 g of silica gel (type Si60, Merck 9385,0.040-0.063 mm) in methylene chloride/methanol/water 70:30:5 (20 mlfractions). Fractions 47-80 are combined and concentrated by evaporationunder a high vacuum at 30°. The residue (3.10 g; greasy crystals) issuspended in 30 ml of diethyl ether. The suspension is stirred for 1/2hour at room temperature. The resulting crystals are then filtered offwith suction and washed with ether. The filter material is taken up in amixture of 15 ml of water and 20 ml of ethyl acetate, the pH is adjustedto 1 with 4N hydrochloric acid and the ethyl acetate phase is separatedoff. After washing the ethyl acetate phase with a total of 20 ml ofwater, all the ethyl acetate phases are combined, dried over sodiumsulfate, filtered and concentrated by evaporation under a high vacuum at30° . The residue (0.41 g) is dissolved in 10 ml of ethyl acetate, and0.437 ml of dicyclohexylamine (Fluka, puriss.) is added to the solutionto give the dicyclohexylammonium salt of2-methyl-2-(tetrahydropyran-4-yloxy)-propionic acid in the form ofcolourless crystals which melt at from 136.6° to 138.8° (sintering from130° ) and can likewise be reacted as described above to form the free2-methyl-2-(tetrahydropyran-4-yl-oxy)-propionic acid.

Alternatively, the dicyclohexylammonium salt of2-methyl-2-(tetrahydropyran-4-yloxy)propionic acid can also be obtainedin accordance with the following process:

Stage 8.1c: 10.21 g (100 mmol) of tetrahydro-2H-pyran-4-oil (Fluka,pract.) are dissolved in 350 ml of absolute 1,4-dioxane (Fluka,puriss.). The resulting solution is heated to 65°. Then 12.0 g (300mmol) of sodium hydride (60% strength, in oil; Fluka, pract.) are addedat 65°. The resulting grey suspension is stirred for 3 hours underreflux and subsequently cooled to 65° again, after which a solution of16.70 g (100 mmol) of α-bromoisobutyric acid (Fluka, pract.) in 150 mlof absolute dioxane is added dropwise over a period of 25 minutes. Theresulting suspension is stirred for a further 3 hours under reflux andthen for 17 hours at room temperature 25 ml of water are then carefullyadded dropwise and the yellow suspension is concentrated to dryness byevaporation under a high vacuum. The residue is taken up in 50 ml ofwater and extracted twice with 100 ml of ethyl acetate each time. Theextracts are then washed once with 50 ml of water. The aqueous phasesare combined, adjusted to pH 1 with 4N hydrochloric acid, sainted withsodium chloride and extracted twice with 50 ml of ethyl acetate eachtime. The extracts are then washed twice with 50 ml of saturated sodiumchloride solution each time. All the ethyl acetate phases are combined,dried over sodium sulfate, filtered and concentrated by evaporationunder a high vacuum. The residue (5.22 g, yellow oil) is purified byflash chromatography (all 25 ml fractions) at 0.4 bar on 500 g of silicagel (type Si60, Merck 9385; 0.040-0.063 mm) in methylenechloride/methanol (9:1; fractions 1-50), methylene chloride/methanol(4:1; fractions 51-150) and methylene chloride/methanol (7:3; fractions151-225). Fractions 16-200 are combined and concentrated by evaporationunder a high vacuum. The residue is stirred with 25 ml of diethyl etherfor 1/4 hour at room temperature. The resulting crystals are filteredoff with suction and washed with diethyl ether. The colourless crystalsare then taken up in 10 ml of water, adjusted to pH 1 with 4Nhydrochloric acid and extracted twice with 20 ml of ethyl acetate eachtime. The ethyl acetate phases are washed twice with 10 ml of water eachtime and then combined, dried over sodium sulfate, filtered andconcentrated by evaporation under a high vacuum. The residue isdissolved in 10 ml of ethyl acetate, and 0.317 ml of dicyclohexylamineis added to give the dicyclohexylammonium salt of2-methyl-2-(tetrahydropyran-4-yloxy)-propionic acid in the form ofcolourless crystals which melt at from 136.5° to 138.8° (sintering from130° ).

Example 9: Tablets each comprising 20 mg of active ingredient, forexample one of the compounds of formula I described in the precedingExamples, are prepared in customary manner with the followingcomposition:

    ______________________________________                                        Composition                                                                   ______________________________________                                        active ingredient                                                                              20 mg                                                        wheat starch     60 mg                                                        lactose          50 mg                                                        colloidal silica  5 mg                                                        talc              9 mg                                                        magnesium stearate                                                                              1 mg                                                                         145 mg                                                       ______________________________________                                    

Preparation: The active ingredient is mixed with a portion of the wheatstarch, with the lactose and the colloidal silica and the mixture isforced through a sieve. A further portion of the wheat starch is madeinto a paste with 5 times the amount of water on a water bath and thepowder mixture is kneaded with the paste until a slightly plastic masshas been formed.

The plastic mass is pressed through a sieve having a mesh size ofapproximately 3 mm and dried, and the resulting dry granules are forcedthrough a sieve again. The remaining wheat starch, the talc and themagnesium stearate are then admixed and the mixture is compressed toform tablets each weighing 145 mg and having a breaking notch.

Example 10: Anti-tumour activity ofN-[0-(tetrahydropyran-4-yl)-D-lactoyl]-staurosporin in vivo:

The substance is formulated as follows: 12.5 mg of active ingredient aredissolved in 0.25 ml of dimethyl sulfoxide and mixed with 50 μof Tween80.4.7 ml of a 0.9% sodium chloride solution are then added and thewhole is mixed thoroughly.

Female Balb/c hairless mice with s.c. transplanted human bladder tumoursT24 are used to determine the anti-tumour activity. With the animalsunder peroral forene narcosis, approximately 25 mg of a solid tumour areplaced under the skin on the animals' left flank on day 0 and the smallincised wound is closed by means of suture clips. On day 6 after thetransplantation, the mice are divided at random into groups of 6 animalsand treatment commences. The treatment is carried out for 15 days withthe administration once daily of the various doses. The tumours aremeasured twice a week with a slide gauge and the volume of the tumoursis calculated. The results are compiled in the following Table in which"dose" is the daily dose, "admin." is the mode of administration,"exper." is experiment and "T/C %" is the percentage quotient of thevalues in the treated mice and the untreated control mice. The smallerthe quotient, the more effective is the dose administered.

    ______________________________________                                                              average tumour volume                                   dose                  [T/C %]                                                 [mg/kg]  admin.       Exper. 1 Exper. 2                                       ______________________________________                                        6.25     p.o.         15       12                                             3.13     p.o.         17       14                                             1.56     p.o.                  31                                             0.78     p.o.                  58                                             3.00     i.p.          9       11                                             1.50     i.p.         14       19                                             0.75     i.p.                  32                                             0.38     i.p.                  62                                             0.19     i.p.                  74                                             ______________________________________                                    

Example 11: Determination of the maximum tolerated dose (MTD) ofN-[0-(tetrahydro-pyran-4-yl) -D-lactoyl]- staurosporin

3 female Balb/c mice per dose are treated i.p. or p.o. withN-[O]-(tetrahydropyran-4-yl)-D-lactoyl]-staurosporin in dimethylsulfoxide between 80/sodium chloride solution (see Example 10 forformulation). The dose is increased until animals succumb within 7 days.

MTD (p.o.): 62.5 mg/kg

MTD (i.p.): 31.25 mg/kg

What is claimed is:
 1. AnN-(tetrahydropyran-4-yloxy-alkanoyl)-staurosporin derivative of formulaI ##STR7## wherein the depicted configurations are intended toillustrate only the relative stereochemistry, whereas the absolutestereochemistry corresponds to that of staurosporin, and whereinR₁ ishydrogen, hydroxy, lower alkoxy or oxo, R₂ is hydrogen or C₁₋₄ alkyl andR₃ is hydrogen or C₁₋₄ alkyl.
 2. A compound according to claim 1 offormula I, wherein R₃ is hydrogen.
 3. A compound according to claim 1 offormula I, wherein R₁ is hydrogen, hydroxy or oxo, R₂ is hydrogen orC₁₋₄ alkyl and R₃ is hydrogen or C₁₋₄ alkyl.
 4. A compound according toclaim 1 of formula I, wherein R₁ is hydrogen or oxo, R₂ is hydrogen orC₁₋₄ alkyl and R₃ is hydrogen.
 5. A compound according to claim 1 offormula I, wherein R₁ is hydrogen or oxo, R₂ is hydrogen or methyl andR₃ is hydrogen.
 6. A compound according to claim 1 of formula I that hasthe (D)-configuration at the C-R₂ atom when R₂ is different from R₃. 7.N-[O -(tetrahydropyran-4-yl)-D-lactoyl]-staurosporin according toclaim
 1. 8. N-[2-(tetrahydropyran-4-yloxy)-acetyl]-staurosporinaccording to claim
 1. 9. A compound according to claim 1 of the formulaI selected fromN-[O-(tetrahydropyran-4-yl)-D-lactoyl]-7-oxo-staurosporin,N-[O-(tetrahydropyran-4-yl)-L-lactoyl]-staurosporin, N-[O-(tetrahydropyran-4-yloxy)-acetyl]-7-oxo-staurosporin,N-[2-(tetrahydropyran-4-yloxy)-acetyl]-7-hydroxy-staurosporin, and N-[2-methyl-2-(tetrahydropyran-4-yloxy) -propionyl]- staurosporin.
 10. Apharmaceutical composition for administration to a warm-blooded animalcomprising a compound of formula I according to claim 1 in an amount offrom about 1 mg to about 1500 mg/day/70 kg of warm-blooded animal in 1-3divided doses.
 11. A pharmaceutical composition for the treatment ofprotein kinase C dependent proliferative disorders comprising an amounteffective in inhibiting protein kinase C of a compound of formula Iaccording to claim 1 together with a pharmaceutical carrier.
 12. Apharmaceutical composition for the treatment of platelet-derived growthfactor kinase dependent proliferative disorders comprising an amounteffective in inhibiting platelet-derived growth factor kinase of acompound of formula I according to claim 1 together with apharmaceutical carrier.
 13. A method of treating protein kinase Cdependent proliferative disorders in a warm-blooded animal requiringsuch treatment which comprises administering to said animal a doseeffective in inhibiting protein kinase C of a compound of formula Iaccording to claim
 1. 14. A method of treating platelet-derived growthfactor kinase dependent proliferative disorders in a warm-blooded animalrequiring such treatment which comprises administering to such animal adose that is effective in inhibiting platelet-derived growth factorkinase of a compound of formula I according to claim 1.